Most of bispecific antibodies (BsAbs) are artificially manufactured to bind two different targets simultaneously rather than generally produced in nature. A double targeting ability provides BsAbs with new applicable field, which has not been managed by monopecific antibodies (MsAbs). Special interest in therapeutic purposes is the provoking possibility, such that BsAbs (1) reliably recruit immune cells into the proximity of target cells, (2) inhibit or activate two distantly apart signaling pathways in target cells to create synergetic effects, and (3) deliver radiation-induced therapeutic substances, medical drugs, toxins or signaling molecules in a specific- and regulatory manner.
BsAbs are generally utilized for delivering T cells to tumor cells in a MHC-independent way, mediating a linkage between cell surface antigens of tumor cells and CD3-TCR complex of cytotoxic T cells (FIG. 1). Catumaxomab(Removab®), rat-mouse hybrid monoclonal antibody, in FIG. 1 is used to treat malignant ascites, which is called “Trifunctional antibody.”
Complete chain association should occur at two different levels, in order to produce minimally modified full-length IgG-like BsAbs without any chain association problem. (1) Two heavy chains should be heterologous bispecific, and (2) two light chains (LC) should pair correctly with their respective heavy chains.
Chain association issues should be solved to produce BsAbs in a trustworthy method. As shown in FIG. 2, combination of two heavy chains and two light chains generates 10 different forms of antibody chimera. Among them, only one is a correct BsAb, and the rest are worthless Chimera. This chain association issue reduces production yield of correct BsAb to at least 10 times in industry fields, and causes various problems with difficulties in isolating BsAbs from other chimera. Therefore, many pharmaceutical companies spend a lot of resources and make efforts to develop and obtain technology for producing BsAbs in a direct and reliable way.
Many various BsAb-related techniques (45 different formats) have been developed. These techniques are classified into 4 categories based on the structure. First, heterologous bispecification of heavy chains by various methods comprising structural complementarity kown to Knob-into-Hole or simply KiH, electrostatic steering effect, or CH3 domain shuffling (called to SEEDbody™); second, various antibody fragment formats such as Diabody™, BiTE™ and DART™; third, technology using one or more functional domains combined with intact antibodies, such as Modular Antibody™, Zybody™, dAbs™ and DVD-IG™; and fourth, techniques adopting full length IgG-like scheme as Duobody™ (Fab-Arm Exchange), CrossMab™, Azymetric™, and kI body™ have been developed.
Out of them, Zymeworks through the United States Patent Application No. 2013-892198, claiming a patent for the structure of heteromultimer immunoglobulin chains having mutations in Fc domain, showed that the antibodies of the heterologous multimeric structure could be made by modifying cysteine residues involved in disulfide bonds with charged amino acids.
However, any patent above has not disclosed such a technology that a modified amino acid pair selected from the portion of the hydrophobic interaction induces to selectively couple each other by the electrostatic interaction. The inventors have completed the present invention by confirming that heterologous bispecification takes place more selectively when one pair of amino acids involved in hydrophobic interaction are modified to an acidic amino acid and a basic amino acid, respectively.